Oncolytic virus therapy

ABSTRACT

A method of treating a human subject with cancer is disclosed. A pharmaceutical composition is administered to the subject, the pharmaceutical composition comprising human leukocytes and a replication-competent oncolytic virus in suspension in a physiologically acceptable solution. Alternatively the pharmaceutical composition comprises human leukocytes or platelets infected with an oncolytic virus.

This application claims the benefit of U.S. Provisional Application No.60/290,051, filed May 11, 2001, the content of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

“Treatment of Neoplasms with Viruses” (WO 00/62735) relates to a methodof administering viruses that are able to replicate and kill neoplasticcells with a deficiency in the IFN-mediated antiviral response. Onespecific aspect of this patent involves the “systemic” administration ofsuch viruses.

Paramyxoviruses are known to interact with erythrocytes and agglutinatethem. They are reported to elute from erythrocytes with lower efficiencythan influenza viruses. Howe, C. and Lee, L. T. (Adv. Virus Res.17:1-50, 1972). Furthermore, the specific inhibition of hemagglutinationof erythrocytes caused by paramyxoviruses through neutralization byantibodies specific to viral coat proteins is a well understoodphenomenon.

Schirrmacher et al (Int. J of Oncology 16:363-373, 2000) have shown thatNDV will activate mouse macrophages for anti-tumor activity. Theexperimental results appearing below demonstrate that NDV does notpreferentially bind to mouse leukocytes when added to mouse whole blood.

Bonina et al (Giorn. Batt. Virol. Immun., LXXVIII, 254-261, 1985) showthat human macrophages could support the growth of NDV.

Woodruff et al (Cellular Immunology 5:296-306, 1972) and Woodruff andWoodruff (J of Immunology 112 (6);2176-2183, 1974) found that NDVagglutinates rat, mouse and human lymphocytes in vitro.

Faden et al (Blood, 58:221-7,1981) showed that NDV will bind to humanneutrophils in vitro.

None of the above indicate any preferential binding of NDV to leukocytesover erythrocytes.

The literature describes the binding of NDV to red blood cells (usuallychicken). As NDV is not pathogenic in man it is a surprising result tofind that NDV binds preferentially to the white blood cell component ofhuman blood.

The literature indicates that the binding of NDV to cells occurs throughthe interaction of the Neuraminidase of the viral HN protein with sialicacid residues attached to cell surface proteins. Human erythrocytes havea very high density of sialic acid residues attached to surfaceproteins. The ratio of erythrocytes to leukocytes in human blood isapproximately 1000 to 1. Thus, it is especially surprising that NDVbinds to the leukocyte fraction instead of the much more numerouserythrocytes.

SUMMARY OF THE INVENTION

This invention provides a method of treating a human subject withcancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human leukocytes and areplication-competent oncolytic virus in suspension in a physiologicallyacceptable solution, wherein the virus binds specifically to theleukocytes; and the ratio of plaque-forming units of the virus to numberof leukocytes in the composition is at least 1:100, thereby treating thesubject.

This invention provides a method of treating a human subject withcancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human cells infected with anoncolytic virus in suspension in a physiologically acceptable solution,wherein the cells are leukocytes or platelets, thereby treating thesubject.

This invention provides the use of a pharmaceutical composition to treata human subject with cancer or in the manufacture of a medicament forthe treatment of cancer, the pharmaceutical composition comprising: (a)human leukocytes and a replication-competent oncolytic virus insuspension in a physiologically acceptable solution, wherein the virusbinds specifically to the leukocytes and the ratio of plaque formingunits of the virus to number of leukocytes in the composition is atleast 1:100; or (b) human cells infected with an oncolytic virus insuspension in a physiologically acceptable solution, wherein the cellsare leukocytes or platelets. Uses (a) and (b) are linked in thatpracticing use (a) generally involves practicing use (b) since thereplication-competent oncolytic virus will generally infect theleukocytes.

This invention is based, in part, on the finding that an oncolytic virussuch as NDV binds to leukocytes and platelets. NDV binds leukocytespreferentially compared to erythrocytes. Tumors involve inflammatoryprocesses. Therefore leukocytes to which an oncolytic virus is bound orwhich are infected with an oncolytic virus are a particularly effectivemeans of delivering oncolytic viruses.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “human cells” means cells isolated from a human,or cultured cells that have been derived from cells isolated from ahuman and/or whose nucleic acid component has been altered by, forexample, immortalization, irradiation or recombinant means. “Humanleukocytes” and “human platelets” are human cells that are leukocytesand platelets, respectively. Except where otherwise specified orrequired by the context the terms “cells” or “human cells” refer toleukocytes and/or platelets.

As used herein the term “plaque-forming unit” (pfu) means one infectiousvirus particle.

As used herein the term “multiplicity of infection” (MOI) means thenumber of infectious virus particles added per cell.

As used herein the term “clonal virus” means a virus derived from asingle infectious virus particle and for which individual molecularclones have significant nucleic acid sequence homology. For example, thesequence homology is such that at least eight individual molecularclones from the population of virions have a sequence homology greaterthan 95% over 300 contiguous nucleotides.

As used herein the term “leukocyte virus complex” (LVC) means thecomplex formed when an oncolytic virus is mixed with a leukocyte cellpopulation and the virus has become associated with the cells. The termincludes both cells where the virus is bound to the outside of the celland cells which are infected by the virus.

As used herein a virus is said to “bind(s) specifically” to a given cellif such virus binds to such cell with a greater specificity than suchvirus binds to erythrocytes. The terms bind(s) specifically andspecifically bind(s) are used interchangeably.

As used herein “NDV” is an abbreviation for Newcastle Disease Virus.

As used herein the term “replication-competent” virus refers to a virusthat produces infectious progeny in cancer cells.

As used herein the transitional term “comprising” is open-ended. A claimutilizing this term can contain elements in addition to those recited insuch claim.

In accordance with this invention the human cells can be derived fromany source. They can be donated by someone other than the subject.However if feasible it is generally preferred to use cells donated bythe subject, for safety reasons. Optionally cells isolated from thedonor can first be grown in culture and the cultured cells are alsoconsidered to be cells of the donor from which they were derived.Examples of cultured cells that can be utilized in accordance with thisinvention include immortalized human leukocyte cell lines. Suitable celllines are publicly available from sources such as the American TypeCulture Collection, for example U-937 (ATCC No. CRL-1593.2) and KG-1(ATCC No. CCL-246).

The leukocytes utilized in accordance with this invention (e.g.monocytes, neutrophils and lymphocytes including tumor-infiltratinglymphocytes) can be active or inactive. Techniques for inactivatingleukocytes include irradiation.

The cells utilized in accordance with this invention can be isolated(for example by leukopheresis in the case of leukocytes). However it isnot necessary to isolate the cells and whole blood can be used instead,in which case the pharmaceutical composition comprises the oncolyticvirus suspended in whole blood or whole blood containing leukocytesand/or platelets infected with the virus. Optionally the leukocytes orplatelets are first isolated from whole blood, mixed or infected withthe virus and then added back to the other blood components.

In different embodiments of this invention the leukocytes are selectedfrom monocytes, neutrophils and lymphocytes. In a more specificembodiment of this invention the leukocytes are tumor-infiltratinglymphocytes (TILs). TILs may be prepared for example by the methoddescribed in Rabinowich, H., et al., (Cancer Res. 47: 173-7, 1987).

In accordance with this invention the oncolytic virus utilized can be oflow (lentogenic), moderate (mesogenic) or high (velogenic) virulence.The level of virulence is determined in accordance with the Mean DeathTime in Eggs (MDT) test. (Alexander, “Chapter 27: Newcastle Disease” inLaboratory Manual for the Isolation and Identification of AvianPathogens, 3^(rd) ed., Purchase, et al. eds. (Kendall/Hunt, Iowa), page117.) Viruses are classified by the MDT test as lentogenic (MDT>90hours); mesogenic (MDT from 60-90 hours); and velogenic (MDT<60 hours).

In an embodiment of this invention the virus is a clonal virus.

Referring to the method or use in which the pharmaceutical compositionutilized comprises leukocytes and oncolytic virus in suspension, in anembodiment of such method the ratio of plaque-forming units of the virusto number of leukocytes in the composition is at least 1:1. Generally itis preferred that the leukocytes be saturated with active virusparticles. In the case of NDV saturation is achieved at a 200:1 ratio ofplaque-forming units of the virus to number of leukocytes. Accordinglyin an embodiment of this invention the virus is NDV and the ratio ofplaque-forming units of the virus to number of leukocytes in thecomposition is from about 1:1 to about 200:1, and preferably is about200:1.

In the method or use described above in which the pharmaceuticalcomposition utilized comprises cells infected with an oncolytic virus,in an embodiment of such method the infected cells are at leastone-tenth of one percent (0.1%) of the total number of leukocytes andplatelets in the composition, more preferably at least thirty percentand most preferably about one hundred percent. The virus utilized can bereplication incompetent although preferably it is replication competent.

In an embodiment of this invention the oncolytic virus is selected fromthe group consisting of a Newcastle Disease Virus (NDV), a Mumps Virus,a Measles Virus, a Vesicular Stomatitis Virus, a Para-influenza Virus,an Influenza Virus, an Adenovirus, a Herpes I Virus, a Vaccinia Virus,and a Reovirus. In a more specific embodiment a Newcastle Disease Virusstrain of moderate virulence can be utilized.

The skilled clinician can determine the optimal amount of thecomposition to be administered in each case. Typically when the cellsare leukocytes the effective amount is a daily dosage of the compositioncontaining from 6×10⁶ to 6×10¹⁰ leukocytes per square meter of patientsurface area, for example about 6×10⁷ leukocytes per square meter ofpatient surface area. When the cells are platelets the effective amountis typically a daily dosage of the composition containing from 10⁹ to10¹¹ platelets per square meter of patient surface area, for exampleabout 10¹¹ platelets per square meter of patient surface area.

The daily dosage of the composition can be administered to the subjectin multiple administrations in the course of a single twenty-four hourperiod in which a portion of the daily dosage is administered at eachadministration. More preferably the daily dosage is administered in asingle administration. In an embodiment of this invention the dailydosage of the composition is administered to the subject at a frequencyof from one to seven times (i.e. on each of from one to seven days) in aone-week period.

In accordance with this invention, any conventional route ofadministration is suitable for administering the pharmaceuticalcomposition. For example the composition can be administeredintravenously, intratumorally, intraperitoneally or intravesicularly(kidneys). In the case of intravenous administration it is convenient ifthe volume of the composition administered is from twenty-fivemilliliters to one liter. In the case of intratumoral administration itis convenient if the volume of composition administered is from onehundred microliters to ten milliliters per tumor mass. In the case ofintraperitoneal administration it is convenient if the volume ofcomposition administered is up to two liters. In the case ofintravesicular administration it is convenient if the volume ofcomposition administered is up to seventy-five milliliters, preferablyfrom fifty to sixty milliliters. Depending on the amount of pfus ofvirus and cells to be administered the concentration of the compositioncan be varied to achieve the desired volume. When the cancer is a solidtumor the composition can be administered by any of the routes givenabove, for example intravenously or intratumorally. When the cancer isother than a solid tumor (e.g. leukemia) the composition is notadministered intratumorally and instead can be administered by the otherroutes given above, for example intravenously.

The invention will be better understood by reference to the followingexamples which illustrate but do not limit the invention describedherein. In the following examples the NDV used was a triple-plaquepurified attenuated (moderately virulent) version of the MK107 strain ofNewcastle Disease Virus, described more fully in international patentpublication WO 00/62735, published Oct. 26, 2000 (Pro-Virus, Inc.).

EXAMPLES Example 1 Binding of NDV to Human Blood Cells

Purpose: Investigate the binding of NDV to human blood cells in order todetermine which cell types bind the virus.

Materials: Reference lot RL-004 NDV; Antibody Mab2F12 (3.5 mg/ml) israised to NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2%fetal bovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10×FACS Lysing Solution, Cat349202, lot#82026

Method: Human whole blood was collected in citrate tubes (3.2%, 0.105 M,Becton Dickinson #366415). Approximately 4 ml from each of 2 tubes waspooled and kept at room temperature until use. Cells were counted usingthe trypan blue exclusion method and a hemocytometer. NDV lot numberRL-004 (1.3E+10 PFU/ml) was used to infect the cells at MOIs(Multiplicity of Infection, expressed as PFU/cell) of 0.2, 0.05 and 0.02(along with a negative control of no added virus). After virus wasadded, tubes were incubated at 37° C. for 30 minutes. Gentle mixing ofthe tubes to keep cells in suspension was performed at 3 intervalsduring the incubation period.

The samples were washed twice by adding 2 ml of cold PSB, centrifugingfor 5 minutes, 4° C., 2000 rpm, and aspirating the solution away fromthe cell pellet. The PSB was removed each time by aspiration. Thevolumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described.The goat anti-mouse—PE reporter antibody was added to each sample byadding 1 ml of a 12 μg/ml solution of this antibody. The samples wereagain incubated for 30 minutes on ice and washed as described above. Foranalysis of the leukocyte fraction, 100 μl of each sample was incubatedwith 3 ml of 1×FACS Lysing Solution for 6 minutes at room temperature.The samples were centrifuged and aspirated as before. The cell pelletswere re-suspended in 0.5 ml of PSB. For erythrocyte analysis, 1.5 μl ofeach sample was added to 2.0 ml of PSB. Samples analysis was performedwith a Becton Dickinson FACSCalibur flow cytometer. Forward and sidescatter parameters employed linear settings and FL2 detection ofphycoerythrin employed logarithmic settings.

Results: The results of the experiment shown in Table 1 indicate thatNDV binds preferentially to the leukocyte fraction of human whole blood.At an MOI of 0.05 (to whole blood), 46% of the leukocytes are positivefor NDV compared to 0% of the erythrocytes. At an MOT of 0.2 NDV ispresent on 89% of the leukocytes while bound to only 15% of theerythrocytes. Note that MOI's of 0.05 and 0.2 are approximately 50 and200 to the leukocytes if the presence of the erythrocytes is discounted.The small amount of NDV binding to erythrocytes at the higher MOI mayreflect low affinity binding to sialic residues on proteins present onthe surface of these cells.

TABLE 1 % Cells Positive for NDV Binding MOI Erythrocytes Leukocytes0.05 0 46 0.2 15 89

It is generally appreciated that Paramyxoviridae interact witherythrocytes and produce haemagglutination of the erythrocytes (see How,C. and Lee, L. T., “Virus-Erythrocyte Interactions”). Thus, it iscommonly believed that the major cell type involved in the binding ofthese viruses, including NDV, are the erythrocytes. The binding of NDVto cells is thought to occur through the interaction of theNeuraminidase activity of the viral HN protein to sialic acid residuesattached to cell surface proteins. Human erythrocytes have a very highdensity of sialic acid residues attached to surface proteins for thepurpose of keeping the cells in solution in the blood. As the ratio oferythrocytes to leukocytes in human blood is approximately 1000 to 1, itis especially surprising that NDV binds to the leukocyte fractioninstead of the vastly more numerous erythrocytes.

Example 2 NDV Associates with Leukocytes in the Presence of NDVNeutralizing Antibody

Introduction/Background: The purpose of this experiment was to assessthe ability of NDV to bind to leukocytes in the presence or absence ofNDV neutralizing antibody. Human clinical patient 521 receivedapproximately 27 treatment courses of NDV therapy prior to having wholeblood drawn for this experiment. Patient 521 displayed significantlevels of NDV neutralizing antibodies as detected by plaqueneutralization and micro-neutralization assays.

Methods: Human whole blood from a naive donor and patient 521 werecollected in citrate tubes (3.2%, 0.105M, Becton Dickenson #366415). Thetubes from each donor were kept at room temperature. Whole blood wasdivided into aliquots and spiked with NDV (lot number RL-005). NDV wasspiked at MOIs (multiplicity of infection, expressed as PFU/cell) of0.2, 0.02 and 0.002 (along with a negative control of no added virus).After virus was added, tubes were incubated at room temperature for 30minutes. Plasma and leukocytes were isolated from the spiked samples bygradient centrifugation using polymorphprep (Nycomed, Inc.). Plasma wascarefully removed from the top of the gradient. Two Leukocyte bands (apolymorphonuclear cell band and a mononuclear cell band) were collectedand placed in 0.5×DMEM. Cells were pelleted and resuspended in fullstrength DMEM. Cells were washed several times to remove the separationmedia. Leukocytes in each aliquot were enumerated using a CoulterCounter. Known numbers of leukocytes or volumes of plasma wereco-cultured or inoculated onto monolayers of HT1080 human fibrosarcomacells (ATCC, CCL-121) in 25 cm² tissue culture flasks (Corning). HT1080cells are highly sensitive to cytolysis by NDV. Monolayers wereevaluated qualitatively over several days for the presence of CPE(cytopathic effect). Flasks which exhibited CPE were consideredpositive.

Results: As shown in Table 2, for both the naive donor and patient 521,at 300,000 leukocytes per flask for all three MOI spikes, both flaskstested were positive for infectivity by NDV. For the naïve donor, allflasks were positive at the 3000 cell level and all but the lowest MOIspike was positive at the 30 cell level. At the 3000 cell level forpatient 521, 1 of 2 flasks were positive at the 0.02 MOI and 0 of 2 atthe 0.002 MOI. At the 30 cell level for patient 521, 1 of 2 flasks werepositive at the 0.2 MOI, all other flasks at this cell level werenegative.

The plasma data show that in the naive patient, virus could be recoveredfrom the plasma at all MOIs tested (see Table 3). However, no infectiousvirus was recovered from patient 521 plasma which contained neutralizingantibody.

Discussion: The results show that the number of leukocytes associatedwith infectious virus was reduced in patient 521 compared to the naïvedonor. However, virus was still able to bind to leukocytes in thepresence of neutralizing antibody. This binding was shown to occur evenat low MOI (0.002), which is similar to the MOI for patient dosing.

NDV elicits a humoral immune response which results in the production ofneutralizing antibody. Binding of virus to leukocytes may allow thevirus to be protected from the neutralizing antibody. This is anadvantage over free virus, which is exposed to the neutralizing antibodyand rendered non-infectious.

It can be concluded from these data that using leukocytes as a vehicleto deliver virus to tumors is an advantage over free circulating viruswhich would be rendered non-infectious by neutralizing antibody inpatients that have generated an immune response (ie. Patients which havereceived multiple doses of NDV).

TABLE 2 Evaluation of Leukocytes for the Presence of Bound InfectiousNDV Number of Positive Cultures After Leukocytes are Placed inCo-Culture with a Monolayer of HT1080 NDV Spike Cells in a T25 Flask(MOI) 300,000 Cells 3000 Cells 30 Cells Naïve Donor 0.2   2* 2 2 0.02  22 2 0.002 2 2 0 No Spike (Ctl.) 0 0 0 Patient 521 0.2   2* 2 1 0.02  2 10 0.002 2 0 0 No Spike (Ctl.) 0 0 0 *Number of positive flasks per 2flasks tested.

TABLE 3 Evaluation of Plasma for the Presence of Infectious NDV SampleNaïve Donor Patient 521 0.2 MOI Spike 1 0 0.02 MOI Spike 1 0 0.002 MOISpike 1 0 No Spike (Control) 0 0 N = 1 flask

As NDV is not pathogenic in man it is a surprising result to find thatNDV binds preferentially to the white blood cell component of humanblood.

Example 3 Preparation of Human Leukocyte/NDV Complex

Human Leukocytes (5×10⁺⁸ cells) are prepared by leukopheresis or bygradient centrifugation employing POLYMORPHPREP (Nycomed, Inc.) usingthe manufacturer's instructions. The cells are washed twice in sterile1×PBS at room temperature and brought to a volume of 10 ml in the samebuffer. The cells are mixed with 1×10⁺¹⁰ pfu of NDV (added aseptically)and allowed to sit for 30 minutes (with further brief mixings at 10 and20 minutes). The cells are centrifuged for 5 minutes at 1500 rpm and thePBS removed. The cells are washed with 20 ml of 1×PBS and centrifugedagain. The cell pellet is diluted to 10 ml for injection.

Example 4 Treatment of Human Tumor Xenografts (<10 mm and >5 mm) inAthymic Mice with Human Leukocyte/NDV Complex

Athymic mice are injected intradermally with 10 million human tumorcells. After tumors reached a size range of between 5 and 10 mm, asingle injection of 8×10⁺⁶ cells of the Leukocyte/NDV complex describedin Example 3 is given. The effect of LVC on tumor growth is examined forcomplete and partial regressions.

Example 5 Systemic Treatment of Human Tumors with Human Leukocyte/NDVComplex in Patients

Human leukocytes are obtained by leukopheresis from patients. Theleukocyte/NDV complex (LVC) is prepared as described in Example 3. Thecomplex (5×⁺⁸ cells) is returned to the patient by intravenousinjection. Injections of LVC are given at three-day intervals for 21days.

Example 6 Systemic Treatment of Human Tumors with Human DonorLeukocyte/NDV Complex in Patients

Human leukocytes are obtained by leukopheresis from naïve donors. Theleukocyte/NDV complex (LVC) is prepared as described in Example 3. Thecomplex (5×10⁺⁸ cells) is returned to the patient by intravenousinjection. Injections of LVC are given at three-day intervals for 21days.

Example 7 Binding of NDV to Mouse Blood Cells

Purpose: Investigate the binding of NDV to mouse blood cells in order todetermine which cell types bind the virus.

Materials: Reference lot RL-005 NDV; Antibody Mab2F12 (3.5 mg/ml) israised to NDV HN protein; RD PharMingen Stain Buffer (PSB) containing 2%fetal bovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10×FACS Lysing Solution, Cat349202, lot#82026.

Method: Mouse whole blood was collected in citrate tubes (3.2%, 0.105M,Becton Dickinson #366415). Cells were counted using the trypan blueexclusion method and a hemocytometer. NDV lot number RL-005 (4.2E+10PFU/ml) was used to infect the cells at MOIs (Multiplicity of Infection,expressed as PFU/cell) of 0.2, 1 and 3 (along with a negative control ofno added virus). After virus was added, tubes were incubated at 37° C.for 30 minutes. Gentle mixing of the tubes to keep cells in suspensionwas performed at 3 intervals during the incubation period.

The samples were washed twice by adding 2 ml of cold PSB, centrifugingfor 5 minutes, 4° C., 2000 rpm, and aspirating the solution away fromthe cell pellet. The PSB was removed each time by aspiration. Thevolumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. For analysis ofthe leukocyte fraction, 100 μl of each sample was incubated with 3 ml of1×FACS Lysing Solution for 6 minutes at room temperature. The sampleswere centrifuged as before. The cell pellets were re-suspended in 0.5 mlof PSB. For erythrocyte analysis, 1.5 μl of each sample was added to 2.0ml of PSB. Samples analysis was performed with a Becton DickinsonFACSCalibur flow cytometer. Forward and side scatter parameters employedlinear settings and FL2 detection of phycoerythrin employed logarithmicsettings.

Results: The results of the experiment shown in Table 4 indicate thatunlike the binding of NDV to human blood cells where the viruspreferentially binds to leukocytes (Example 1), NDV binds preferentiallyto the erythrocyte fraction of whole mouse blood and does not bind tothe leukocytes.

TABLE 4 % Cells Positive for NDV Binding MOI Erythrocytes Leukocytes 0.230 1 1 80 1 3 92 1

At an MOI of 0.2 where human leukocytes are mostly positive for thebinding of NDV, mouse leukocytes are negative.

Example 8 Binding of NDV to Rat Blood Cells

Purpose: Investigate the binding of NDV to mouse Rat blood cells inorder to determine which cell types bind the virus.

Materials: Reference lot RL-005 NDV; Antibody Mab2F12 (3.5 mg/ml) israised to NDV HN protein; RD PharMingen Stain Buffer (PSB) containing 2%fetal bovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10×FACS Lysing Solution, Cat349202, lot#82026.

Method: Whole blood (1.8 ml) was collected in citrate tubes (3.2%,0.105M, Becton Dickinson #366415) from a Sprague Dawley rat and kept atroom temperature until use. Cells were counted using the trypan blueexclusion method and a hemocytometer. NDV lot number RL-005 (4.2E+10PFU/ml) was used to infect the cells at MOIs (Multiplicity of Infection,expressed as PFU/cell) of 0.05, 0.2, 0.5, 1 and 3 (along with a negativecontrol of no added virus). After virus was added, tubes were incubatedat 37° C. for 30 minutes. Gentle mixing of the tubes to keep cells insuspension was performed at 3 intervals during the incubation period.

The samples were washed twice by adding 2 ml of cold PSB, centrifugingfor 5 minutes, 4° C., 2000 rpm, and aspirating the solution away fromthe cell pellet. The PSB was removed each time by aspiration. Thevolumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. For analysis ofthe leukocyte fraction, 100 μl of each sample was incubated with 3 ml of1×FACS Lysing Solution for 6 minutes at room temperature. The sampleswere centrifuged as before. The cell pellets were re-suspended in 0.5 mlof PSB. For erythrocyte analysis, 1.5 μl of each sample was added to 2.0ml of PSB. Samples analysis was performed with a Becton DickinsonFACSCalibur flow cytometer. Forward and side scatter parameters employedlinear settings and FL2 detection of phycoerythrin employed logarithmicsettings.

Results: The results of the experiment shown in Table 5 indicate thatNDV binds to rat leukocytes at a low MOI (0.02 and 0.05) while it doesnot bind well to the erythrocytes at these MOI's. The pattern appears tobe intermediate between the binding of the virus to human leukocytes towhich it binds preferentially and to mouse leukocytes, to which it doesnot bind.

TABLE 5 % Cells Positive for NDV Binding MOI Erythrocytes Leukocytes0.02  3  7 0.05 12 21 0.2 30 29 1 63 58 3 90 66

Example 9 Binding of NDV to Human Platelets

Purpose: Investigate the binding of NDV to human platelets

Materials: Reference lot RL-005 NDV; Antibody Mab2F12 (3.5 mg/ml) israised to NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2%fetal bovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10×FACS Lysing Solution, Cat349202, lot#82026.

Method:

Isolation of Platelets

Whole human blood was collected in citrate tubes (3.2% 0.105M Citrate,Becton Dickinson #366415). Seven milliliters of whole blood were placedinto a 15 ml polypropylene centrifuge tube. The tube was centrifuged at800 g for 5 minutes at room temperature. Approximately 1.5 ml ofplatelet-rich plasma (PRP) was collected from the top of the centrifugetube. Based on published values for yield, the sample was determined tocontain 8.0E+8 cells/ml. 100% of PRP was infected with NDV (RL-005) atMOIs of 0.1, 10 and 100 for 30 minutes at room temperature.

The samples were washed twice by adding 2 ml of cold PSB, centrifugingfor 5 minutes, 4° C., 2000 rpm, and aspirating the solution away fromthe cell pellet. The PSB was removed each time by aspiration. Thevolumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding0.1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. Sample analysiswas performed with a Becton Dickinson FACSCalibur flow cytometer.Forward and side scatter parameters employed linear settings and FL2detection of phycoerythrin employed logarithmic settings.

Results: The results of the experiment shown in Table 6 indicate thatNDV binds to human platelets. The number of platelets that are positivefor binding of NDV does not increase greatly in the MOI range testedalthough the mean fluorescence does, indicating that more NDV binds tothe positive portion of the platelets as the MOI increases.

TABLE 6 NDV Binding To Human Platelets % Positive Mean MOI PlateletsFluorescence 0 0 10 1 67 258 10 81 616 100 73 1005

Example 10 Binding Hierarchy of NDV to Human Leukocytes

Materials: Reference lot RL-005 NDV; Antibody Mab2F12 (3.5 mg/ml) wasraised to NDV HN protein; BD PharmMingen™ Stain Buffer (PSB) containing2% fetal bovine serum and 0.02% sodium azide, catalog #544656, lotM059394; Rockland goat anti-mouse polyclonal antibody-phycoerythrinconjugate, catalog #710-1832, lot 7367; Becton Dickinson ImmumocytometrySystems™ 10×FACS Lysing Solution Cat. 349202, Lot#82026.

Method: Human whole blood was collected in citrate tubes (3.2%, 0.105M,Becton Dickinson™ #366415). Approximately 4 ml from each of two tubeswas pooled and kept at room temperature until use. Cells were countedusing the trypan blue exclusion method and a hemocytometer. NDV lotnumber RL-004 (1.3E+10 PFU/ml) was used to infect 100 μl samples ofwhole blood at MOIs (Multiplicity of Infection, expressed as PFU/cell)of 0, 0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 to the whole blood. Aftervirus was added, samples were incubated at 37° C. for 30 minutes.

The samples were washed twice by adding 2 ml of cold PSB, centrifugingfor 5 minutes, 4° C., 2000 rpm, and aspirating the solution away fromthe pellet. The volumes of each sample were adjusted to 100 μl with PSB.Monoclonal antibody Mab2F12 was added to each sample by adding 2 μl of asolution made in PSB containing 1 μg of the antibody. The samples wereincubated for 30 minutes on ice and washed twice again as previouslydescribed. The goat anti-mouse—PE reporter antibody was added to eachsample by adding 100 μl of a 12 μg/ml solution of this antibody diluted2.4:1 in PSB. The samples were incubated for 30 minutes on ice andwashed as described above. To prepare the samples for leukocyte cellanalysis, each sample was incubated with 3 ml of 1×FACS Lysing Solutionfor 6 minutes at room temperature, then centrifuged and aspirated asbefore. The cell pellets were re-suspended in 0.5 ml of PSB. Sampleanalysis was performed with a Becton Dickinson FACSCalibur™ flowcytometer. Granulocyte, lymphocyte and monocyte populations were gatedby comparing the forward scatter and side scatter parameters for eachsample. This was used to determine the number of cells positive forbinding the virus and the mean fluorescence value of each of thesepopulations for each sample.

The results (Table 7) indicate that NDV binds preferentially to thethree cell populations. The order of preference is:monocyte>granulocyte>>lymphocyte. Furthermore, as shown from the meanfluorescence data, the monocyte population binds substantially more NDVthan does the granulocyte population, and binds much more virus thandoes the lymphocyte cell population.

TABLE 7 % Positive Cells Mean Fluorescence MOI Monocyte GranulocyteLymphocyte Monocyte Granulocyte Lymphocyte 0 2 2 2 13 19 7 0.005 80 6819 65 20 9 0.01 92 97 39 134 35 11 0.02 99 100 54 219 66 16 0.05 98 10072 314 113 22 0.10 99 100 84 448 154 31 0.20 100 100 96 692 251 69

1. A method of treating a human subject with cancer, comprisingadministering to the subject an amount of a pharmaceutical compositioneffective to treat the subject, the pharmaceutical compositioncomprising human leukocytes and a replication-competent oncolytic virusin suspension in a physiologically acceptable solution, wherein thevirus binds specifically to the leukocytes; and the ratio ofplaque-forming units of the virus to number of leukocytes in thecomposition is at least 1:100, thereby treating the subject: wherein theleukocytes comprise an immortalized cell line.
 2. The method of claim 1,wherein the immortalized cell line is U-937 or KG-1.
 3. A method oftreating a human subject with cancer, comprising administering to thesubject an amount of a pharmaceutical composition effective to treat thesubject, the pharmaceutical composition comprising human leukocytes anda replication-competent oncolytic virus in suspension in aphysiologically acceptable solution, wherein the virus bindsspecifically to the leukocytes, and the ratio of plaque-forming units ofthe virus to number of leukocytes in the composition is at least 1:100,thereby treating the subject; wherein the pharmaceutical compositioncomprises the oncolytic virus suspended in whole blood.
 4. A method oftreating a human subject with cancer, comprising administering to thesubject an amount of a pharmaceutical composition effective to treat thesubject, the pharmaceutical composition comprising human leukocytes anda replication-competent oncolytic virus in suspension in aphysiologically acceptable solution, wherein the virus bindsspecifically to the leukocytes; and the ratio of plaque-forming units ofthe virus to number of leukocytes in the composition is at least 1:100,thereby treating the subject; wherein the composition is administeredintravesicularly.
 5. The method of claim 4, wherein the volume ofcomposition administered is up to seventy-five milliliters.
 6. Themethod of claim 5, wherein the volume of composition administered isfrom fifty to sixty milliliters.
 7. A method of treating a human subjectwith cancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human cells infected with anoncolytic virus in suspension in a physiologically acceptable solution,wherein the cells are leukocytes or platelets, thereby treating thesubject; wherein the cells comprise an immortalized cell line.
 8. Themethod of claim 7, wherein the immortalized cell line is U-937 or KG-1.9. A method of treating a human subject with cancer, comprisingadministering to the subject an amount of a pharmaceutical compositioneffective to treat the subject, the pharmaceutical compositioncomprising human cells infected with an oncolytic virus in suspension ina physiologically acceptable solution, wherein the cells are platelets,thereby treating the subject.
 10. A method or treating a human subjectwith cancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human cells infected with anoncolytic virus in suspension in a physiologically acceptable solution,wherein the cells are leukocytes or platelets, thereby treating thesubject; wherein the infected cells are at least one-tenth of onepercent of the total number of leukocytes and platelets in thecomposition.
 11. The method of claim 10, wherein the infected cells areat least thirty percent of the total number of leukocytes and plateletsin the composition.
 12. A method of treating a human subject withcancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human cells infected with anoncolytic virus in suspension in a physiologically acceptable solution,wherein the cells are platelets, thereby treating the subject; whereinthe effective amount is a daily dosage containing from 10⁹ to 10¹¹platelets per square meter of patient surface area.
 13. The method ofclaim 12, wherein the daily dosage contains about 10¹¹ platelets persquare meter of patient surface area.
 14. The method of claim 12,wherein the daily dosage is administered in a single administration. 15.The method of claim 12, wherein the daily dosage is administered at afrequency of from one to seven times in a one-week period.